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Probing the Active and Allosteric Sites of Thermotoga maritima ADPGlucose Pyrophosphorylase: Mutational Analyses of the glgC1 and glgC2 Proteins
Author(s) -
Matsui Mikiko,
Wiig Jared,
Lim Shan,
Harake Tala,
Lippert Rachel,
Gatzke Janice,
Reyes David,
Panosian Onnig,
Tennison Brent,
Susoeff Michael,
Nguyen Kelly,
Axelrod Herb L,
Lesley Scott A,
Meyer Christopher R
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.612.4
Subject(s) - allosteric regulation , thermotoga maritima , effector , biochemistry , recombinant dna , amino acid , biology , enzyme kinetics , chemistry , enzyme , escherichia coli , active site , gene
The T. maritima ADP‐glucose pyrophosphorylases (ADPG PPases) glgC1 and glgC2 share only 36.9% identity and ~30% identity with other ADPG PPases. The recombinant His‐tagged and native proteins have been purified to homogeneity and found to be functionally identical. The glgC1 protein was inactive while the glgC2 protein displayed ~15 fold lower apparent affinity for substrates and V max compared to other ADPG PPases and was insensitive to typical effector molecules. In vitro formation of a glgC1/C2 complex resulted in a ~20‐fold stimulation of activity and 2.2‐fold activation by FBP. Co‐expression, crystallization studies and complete kinetic characterization of the complex are in progress. A comprehensive series of altered proteins have been generated and purified to homogeneity to target putative functional amino acids in glgC1 and glgC2. The kinetics analyses of the I24R and D244N glgC2 proteins revealed changes in allostery and activity, respectively. Characterization of other proteins harboring mutations implicated in catalysis and regulation, separately and in complex, is underway. Supported in part by NSF Grant 0448676.